Process for producing l-glutamic acid



United States Patent 3,117,915 PRQCESS FOR PRODUCING L-GLUTAMIC ACID Isamu Shiio, Tokyo, Koji Mitsugi, Kanagawa-ken, Shinichiro Ctsulra, Tokyo, and Toshinao Tsun'oda, Kanagawa-ken, Japan, assignors to Ajin'omoto Kabushiki 3,1l7,9i Patented Jan. 14, 1964 ice advantageously used as carbon source, and urea, ammonia or ammonium salts, such as ammonium chloride, ammonium sulfate, and ammonium carbonate may be used as nitrogen source. However, ammonium acetate maye be used as a material which serves as carbon source Kaisha, Tokyo, Japan, a corporation of Jap 5 and nitrogen source simultaneously. N0 DEW/ing- Filed P 1961, 5913 101,364 In addition to the carbon source and the nitrogen 1 Clfilm- 195-30) source, proper quantities of minerals, and vitamins or This invention relates to a process for producing L- vitamin containing materials and other growth factors glutamic acid. 10 may be added for the completion of the medium. As

This application is a continuation-in-part of applicaexamples of such materials, vitamin B biotin, yeast extion, Serial No. 37,064, filed June 20, 1960, and now tract, corn steep liquor, soybean meal hydrolysate, fish b d d, meal, digested fish meal and casein hydrolysate may be More particularly, the invention contemplates a process mentioned for producing L-glutamic acid comprising cultivation of In the process of the present invention, cultivation is a microorganism such as Brevibacterium roseum, Breviconducted in a medium as described above after adjusting bacterium flavum, Bi'evibacterium lactofermentus and pH to 7.09.0, at 28-33 C. for 2-4 days under shaking Coryizebacterium acetoacidophilum in amedium' conor under stirring and aerationin submerged condition. taining acetic acid or sodium, potassium or ammonium When ammonium acetate is used as both carbon and salts thereof as the sole carbon source together with ninitrogen source, it is necessary to use acetic acid or salts trogen source, and inorganic materials, thereby producing thereof other than ammonium acetate together therewith and accumulating L-glutamic acid abundantly in the culin order to maintain the ratio of carbon to nitrogen withture medium and isolating said L-glutamic acid. in a predetermined range. When pH becomes higher Heretofore, ix-ketoglutaric acid, citric acid, glucose and than 9.0, it may be lowered by adding acetic acid and some others have been used as a carbon source for the when pH becomes lower than 7.0, it may be raised by production of L-glutamic acid by fermentation process. adding ammonia or urea. The production of L-glutamic The numbers of carbon atoms of these compounds used acid is carried out actively when pH is maintained within as a carbon source are always 5-6 or more. It is characthe range of 7 .58.5. When the fermentation is comteristic in the present invention that acetic acid, which is pleted, the broth is sterilized by heating, concentrated a lower aliphatic acid having only two carbon atoms and after removing cells and L-glutamic acid is recovered can be readily, economically and industrially produced by therefrom by any conventional method, for example, by chemical synthesis or salts thereof, is used as a carbon crystallization at the isoelectric point of glutamic acid or source in place of the expensive compounds or natural utilizing ion exchange resin. products as mentioned above. The four species (Brevibacterium roseum ATCC No.

In the present invention, L-glutamic acid having 5 car- 13825, Brevibacterium flavum ATCC No. 13826, Brevibon atoms is' synthesizedbiochemically from acetic acid bacterium lactofermentus ATCC No. 13869, and Coryneor salts thereof having only two carbon atoms contrary bacterium acetoacidophilum ATCC No. 13870) used in to the process as used heretofore in which a compound the following examples of the process of the present inhaving many carbon atoms is converted to L-glutamic vention have bacteriological characteristics as shown in acid. Thus, the present invention is very interesting bio- 40 Table 1 and no microorganism which can be identified chemically and provides a' very advantageous industrial to be the same with any of these four species is found in PIOC5S$ Bergeys Manual of Determinative Bacteriology, 7th edi- For the culture medium in which L-glutamic acid is tion. It is, therefore, considered that the four species produced and accumulated according to the process of belong to new species. the present invention, salts of acetic acid, such as potas- The bacteriological characteristics of these four species sium acetate and sodium acetate, or acetic acid may be are as follows:

TABLE 1 Bacteriological Characteristics of 1; Species Brevibacterium TOSE'ZHTL (ATCC No. 13825 Brevibacterz'um flauum (ATCC No. 13826 Brevibacterium lactofermentus (ATCC No. 13869 C'orynebacterium acetoccidophilum (ATCC No. 13870 1. Form of 0011s-.-.

Short rods, usually single or Short rods, usually single or Short rods, usually single or Agar c0lonics Agar slant 7. Chromogenesis 8. Agar stab 9. Nutrient- Circular, smooth, fairly dry,

entire in the edge.

Linear, butyrous, dryish surface, dull luster rosy, no change of color of the medium.

Cells grown in bouillon also exhibit rosy color. Distinct in synthetic medium (sugar, urea, yeast, extract, 0.8. l, agar etc.)

Growth on the surface and along stub, no growth in the submerged part; aerobic.

No growth on the surface, almost clear, cells are hard deposit and suspend in liquid taking wooly form under shaking.

See footnotes at end of table.

Circular, smooth, dry, entire in the edge.

Linear, butyrous, growth normal, dryish surface, dull luster, yellow.

Cells grown lu bouillon also exhibit yellow color. Distinct in synthetic medium (sugar, urea, yeast, extract, 0.8. 1, agar etc).

Growth on the surface and along stab, no growth in the submerged part, aerobic.

No growth on the surface, almost clear, cells are hard deposit and suspend in liquid taking wooly form under shaking.

two, two, two,

0.5X0.62.0;i 0.5XO.6 2.0[L 0.5 0.9l.4

. Mobility None No N e Spore formation (in do (10 Gram stain Positive Positive Positive Circular, smooth, fairly wet,

entire in the edge, small. Linear, butyrous, active growth, generally opaque,

yellow.

Cells grown in bouillon also exhibit pale yellow color.

Slight growth along stub,

growth on the surface particularly good, aerobic.

No growth on the surface, slightly turbid, little deposit.

Short rods, usually single or two, many variations in the the forms,

0.5X0.(l-2.0p None. D

Positive.

Circular, smooth, dry, entire in the edge.

Linear butyrous, active growth, dryish surface, dull luster, yellow.

TABLE 1Continued Brevibacterium rose'um (ATCC No. 13825 Brevibacten'um flavum (AICC No. 13826 Cory'nebacterium acetoacidophilum (ATOC No. 13870 Brevibacterium Zactofermentus (AICC No. 13869 10. Growth temperature, C. 11. pH for growth- Gelatin liquefaction. l3. Litmus milk C, C, 37C, 42C

None

Not decolorized, only slightly change to alkali.

None

Substantially none Positive Negative do do Nrme . Indole produc- None...

tion. HgS'plOdllC- tion. Nitrate reduction. Hydrolysis of starch. Methyl red test- N egativ 20C, 30C, 37C, 42C

Not decolorized, only slightly change to alkali.

Substantially none Positive 20C, 30C, 37C, C

None

20C, 30C, 37C, 42C. 'H-+ 5.0, 6,0, 7.0, 8.0, 9.0.

Not decolorized, only slightly In a day, acidification coaguchange to alkali.

ration and reduced decolorization of litmus take place. None.

Blackened very slightly, but

considered negative.

Exhibits pale rosy color,

positive.

Negativ None.

Substantially none.

Negative.

Distinctly positive Do.

Voges-Proskauer reaction. NH producon. Catalan:

. Urease Usability of N H4H2P O4. Relation to free oxygen. Sugar fermentation:

Glucose Fructose Galactose. Mannose. Lactose Positive No growth, no growth even with addition of biotin.

Facultatively anaerobic Trehalose. Mannitol Sorbitol- ErythritoL- Xylose Arabinose. Melecit ose Rhamnosc Rafilnose Glycerol Starch Dextrine Inuline Glycogen Melibiose... Cellobiose Esculin a-methylglucoside.

IlllllllllllllllllbVlh';

Illfllllllillllllli i li l i Positive -do- IIIIIIIIIIIIIIIIII No growth, no growth with addition of biot' Facultatively anaerobic Positive.

Positive D o No growth.

Facultatively anaerobic.

even

Facultatively anaerobic 1 ATCC No. 13825 and 13826 were assigned by American Type Culture Collection on June 10, 1960. 2 ATCC No. 13859 and 13870 were assigned by American Type Culture Collection on July 7, 1960. 3 Composition of medium, sugar 0.20.5%, peptone 1.0%, NaCl 0.5%, BCP culture at 30 C. for seven days (5.0 ml. portions are introduced in each of test tubes Durham tube injected).

4 A: Acid produced. 5 No production. 6 G: Gas produced.

Examples of the process of the present invention are described in detail as follows:

Soybean meal hydrolysate 0.1 ml/dl. (Total nitrogen 2.4%

After adjusting pH to 7.5-8.0, the medium was introduced in several flasks of 500 ml. capacity as portions of ml. respectively, and all portions were sterilized by heating at 115 C. for 10 mintues. Brevibacterium' flavum ATCC No. 13826 cultured for 24 hours at 30 C. was inoculated to the medium in the flask and cultured under shaking at 30 C. The microorganism began to grow after about 24 hours from the start of culture and pH gradually rose. Every time when pH became 9.0 or higher, it was adjusted by adding 50% solution of acetic A medium of the following composition was prepared.

Ammonium acetate 2.57% (2.0% as acetic acid). Sodium acetate 4.53% (2.0% as acetic acid). KH PO 0.2%. MgSO -7H O 0.04%. Vitamin B 10 ,ug./dl. Corn steep liquor 0.3%. Soybean meal hydrolysate 0.1 ml./dl. (Total nitrogen 2.4%.)

Fe++ and Mn++ 2 p.p.m. respectively. After adjusting the pH of the medium and sterilizing as in Example 1, separate portions of the medium were inoculated respectively with the following three strains and cultured at 30 C. for 3 days under shaking. During the cultivation, 50% solution of acetic acid was added from time to time for the adjustment of pH, and the total of such added solution of acetic acid in each portion was 2.0 ml.

L-glutamic acid produced was recovered from the broth after completion of fermentation according to conventional process as in Example 1.

The amounts of L-glutamic acid produced by three strains were shown in the following table:

Yield on the basis of acetic acid,

percent Amount of produced L-glutamic acid, g/l.

Strain used Brevibacterium res-cum ATTC No. 13825 Brevibacteriumlartoferme'ntus ATCC No. 13869.

Corynebactcrium acetoacidoohilum ATCC N o.

EXAMPLE 3 A medium of the following composition Was prepared.

After preparing the medium and sterilizing as Example 1, Brevibacterium flavum ATCC No. 13826 Was inoculated to the above medium and cultured at 28 C. to 33 C. for hours under shaking. The pH of the medium Was adjusted between 7.0 and 9.0 during the cultivation, described as in Example 1. The amount of acetate (initial acetate and added acetate for pH adjustment) was 4.9% as acetic acid and 96% of acetate Was consumed.

L-glutamic acid produced amounted to 13 g./l. (yield 26.6% on the basis of acetic acid).

What We claim is:

Process for producing L-glutamic acid characterized by culturing under aerobic condition a microorganism selected from the group consisting of L-glutamic acid producing strains of Brevibacterium roseum, Brevibacterium flavzmz, Brevibacterium lactofermentus and Corynebacterium acetoacidophilum in a medium containing at least one compound selected from the group consisting of acetic acid and sodium, potassium and ammonium salts thereof as the carbon source at pH from 7.0 to 9.0.

References Cited in the file of this patent UNITED STATES PATENTS 3,003,925 Kinoshita et a1 Oct. 10, 1961 OTHER REFERENCES Tomlinson, Journal of Biological Chemistry vol. 209 (1954 pp. 585-609). 

